Dried microorganism cultures and method for producing same

ABSTRACT

Dry microorganism cultures comprising at least one microorganism species in carrier-bound form are present in the form of particles which a) have a particle size of at least about 0.1 mm and b) are compressed; processes for preparing dry microorganism cultures and their use for preparing foodstuffs and feedstuffs are also claimed.

The present invention relates to novel dry microorganism cultures whichcan be used in particular to prepare foodstuffs and feedstuffs, and toprocesses for preparing dry microorganism cultures.

A main area of application of microorganisms, such as bacteria andyeasts, is preparing foodstuffs and feedstuffs. Thus, for example,lactic acid bacteria, such as those of the genus Streptococcus sp. orLactobacillus sp. are used in the preparation of milk products, such assour cream, buttermilk, yogurt, kefir, coumis, curd cheese and in thepreparation of sourdough and for preserving uncooked sausage, such assalami. Lactic acid bacteria, such as those of the genus Lactobacillussp., for instance, are also used in the production of feeds., such assilage.

The microorganism preparations required for preparing foodstuffs andfeedstuffs are usually used in the form of starter cultures. These aregenerally not freshly prepared liquid cultures, but either culturesusually frozen in liquid nitrogen or dry preparations. Dry preparationsare usually preferred, since their transport and storage is technicallyless complex in comparison with frozen preparations.

Very varied types of dry preparations of microorganism cultures areknown from the prior art. Thus, for example, EP-A-0 131 114 describes aLactobacillus preparation where a bacterial cell suspension is appliedto a pulverulent or granulated carrier composition and dried. However,to store the preparation it is necessary to package this in anoxygen-free protective gas atmosphere. DD 840493952 proposesfreeze-drying strains of cultured microorganisms for producing startercultures, packaging them in film and storing them at 279 to 288 Kelvin.JP-A-06/217713 describes the production of special Lactobacillus startercultures by spray-drying. EP-A-0 202 409 proposes subjecting drycultures to a wet granulation, processing the granules to form sphericalparticles and then drying them. In addition, proposals are made in anumber of publications to provide coated dry bacterial preparations (cf.U.S. Pat. No. 3,677,897 for example).

A number of different processes are described in the prior art toproduce dry microorganism preparations. In addition to the freeze-dryingand fluidized-bed drying processes mentioned above, another alternativeproduction method is spray-drying a microorganism suspension. Thus, forexample, Stadhouders, J. et al., in Neth. Milk Dairy J. 23 (1969) 182describes the spray-drying of lactic acid bacteria at 70° C., coupledwith a post-drying step at 27° C. in vacuo. Apparently, preconditioned,i.e. predried, air is not used for the drying. Before the drying, acalcium hydroxide slurry is added to the material to be sprayed. Thecalcium lactate formed during the spray-drying is advantageous, inasmuchas it is said to have a lower hygroscopicity. In other spray-dryingprocesses known from the prior art, bacterial suspensions to which themost varied types of carrier materials have been added in advance aresprayed. Thus, for example according to SU 724113, a bacterialsuspension admixed with dried milk powder, molasses and sodium glutamateis sprayed. According to SU 1616990, a bacterial suspension admixed withthe mineral palygorskite is spray-dried. WO-A-88/06181 describes thespray-drying of a bacterial suspension admixed with clay. JP-A-69/67989describes the spray-drying of yeast cells or bacterial cells which aresuspended in a neutral or slightly acidic solution which comprisesproteins, carboxymethylcellulose, alginate or alginate ester,disaccharides or higher saccharides or polyhydric alcohols.

The dry microorganism preparations which are known to date from theprior art, in particular those preparations which are used for producingfoodstuffs or feedstuffs, have at least one of the followingdisadvantages:

-   -   1) the content of viable microbes per unit weight of the dry        material is very low owing to the production method, so that        large volumes of the dry preparation must be used in the final        application;    -   2) the storage stability is too low, so that the dry        preparations must be used within a few weeks, if storage under        technically complex conditions is impossible;    -   3) the dry preparations have a high dust content, which makes        their processing more difficult;    -   4) the mechanical stability is very low, so that on mixing the        preparation with mineral additives, a finely divided abraded        material is formed and separation of the solid preparation can        be observed;    -   5) the dissolution rate of the dry preparations is not        satisfactory, so that the desired microbiological process for        producing the foodstuff or feedstuff only begins slowly and        unwanted microorganisms are given the possibility of        multiplying, which can lead to considerable losses in quality.

The production processes known to date from the prior art, in particularthe spray-drying processes described to date, are also unsatisfactoryfor at least one of the following reasons:

-   -   1) the processes are technically very complex;    -   2) the microorganism survival rate in drying is too low;    -   3) the moisture content of the dry product is too high.

A first object of the present invention is thus the provision ofimproved dry microorganism cultures which substantially no longer havethe abovementioned deficiencies known from the prior art. In particular,starter cultures which are improved in comparison with the prior art areto be provided. The starter cultures according to the invention areespecially to enable improved production of silage.

A second object of the present invention is the provision of improvedprocesses for producing dry microorganism cultures. In particular, animproved process for spray-drying microorganism cultures should beprovided which enables the production of dry preparations having a highcontent of viable microbes and high storage stability.

The above first object is achieved by providing a dry microorganismculture which comprises at least one microorganism species incarrier-bound form, wherein the culture is present in the form ofparticles which

-   -   a) have a particle size of at least about 0.1 mm and    -   b) are compressed.

The particulate cultures according to the invention are virtually dustfree on account of the chosen particle size. The dust content ispreferably in the range from about 0.01 to 0.05% by weight, based on thetotal weight of the dry culture. This corresponds to a dust index in therange from about 1 to 12 determined gravimetrically by a Casellainstrument.

The particles according to the invention furthermore have a compressed,i.e. compact, structure. This is preferably achieved in their productionby a compression step which is explained in more detail below and hasnot been previously described for dry microorganism preparations. Inthis operation a preliminary product obtained, for example, byspray-drying, freeze-drying or fluidized-bed-drying (such as the powderconcentrate which is obtainable by a spray-drying variant according tothe invention and is described below), which usually has a significantdust content (e.g. a dust index from about 25 to 100), is mechanicallycompressed.

The compression can be performed, for example, by compacting thepulverulent preliminary product under the action of linear forces, e.g.in the range from about 5 to about 25 kN/cm, in particular from about 10to about 15 kN/cm, in conventional compacting apparatuses, for example.However, the preliminary product can also be tabletted under the actionof pressures in the range from about 50 to about 250 MPa, in particularin the range from about 80 to 200 MPa, such as from about 90 to about160 MPa, for instance, in conventional tabletting presses, for example.Particular preference is given to compression by compacting. Inaddition, preference is given to compacting powder concentrates obtainedaccording to the invention by spray-drying.

The provision of microorganism cultures of the type described abovesurprisingly results in the processing, in particular as startercultures, being markedly simplified and, moreover, the mechanicalstability of the particles and thus the danger of separation of starterculture preparations being markedly decreased. Surprisingly, it has alsobeen found that the compression of the pulverulent preliminary productvirtually does not impair product quality with respect to the number ofviable microbes. Rather, owing to the high density achieved, the ingressof air and moisture into the dry preparations according to the inventionis significantly decreased in such a manner that a considerableimprovement in storage stability can be achieved.

Thus, for example, survival rates of 60% and above after storage for oneyear at room temperatures were achieved. Advantageous storage stabilitydata of this type have not been described hitherto.

In particular, the compressed particles can comprise compacted brokenmaterial (i.e. material obtained by comminuting with or withoutclassifying compacted product extrudates) having a diameter of fromabout 0.1 mm to about 2 mm, preferably from 0.3 to 1.25 mm. The diameterhere is a value calculated from the total mass distribution of thecompressed particles and corresponds to the diameter of spheres of equalmass. The edge length of the particles is in the range from about 0.1 to2 mm, in particular from about 0.1 to 1.4 mm.

The compressed particles can, furthermore, be present as tablets of anydesired shape, such as round, polygonal or oval, having a diameter offrom about 2 to 50 mm and a ratio of diameter to thickness of from about1:0.1 to about 10:1, in particular from about 1:1 to about 5:1.

According to a further preferred embodiment of the invention, the drymicroorganism cultures comprise, as further component, an effervescenceadditive, comprising an acid component, such as an organic nonvolatilecarboxylic acid, and a gas-forming component, such as a CO₂-formingcomponent. Effervescence formulations of this type have the particularadvantage of a surprisingly rapid dissolution after application of thestarter culture. As a consequence of this rapid dissolution of thestarter culture in its surrounding medium, rapid multiplication of thestarter culture microorganisms is ensured, as a result of which lossesin quality of the product to be produced using the starter culture canbe avoided surprisingly well.

Preferably, the dry culture compressed according to the inventioncomprises, as carrier, at least one matrix material for embedding themicroorganism cells with or without at least one other additive whichstabilizes the cells.

The carrier used in the dry cultures according to the inventioncomprises at least one matrix component added as coformulant prior tothe drying to usually freshly grown microorganisms, selected from mono-,oligo- and polysaccharides, polyols, polyethers, polymers, such as CMCor PVP, oligo- and polypeptides, from natural sources, such as milk,meat or cereals, derived substances or mixed substances, such as sweetwhey powder, wheat semolina bran, peptone, alginates, mineral compounds,or mixtures of such matrix substances. In addition, additives having astabilizing action can be added together with the matrix substance orlater, for example antioxidants, such as α-tocopherol or ascorbic acid,or mixtures thereof. Furthermore, a stabilizing action can be exerted byother substances, which are selected from inorganic salts, such asalkali metal chlorides or alkaline earth metal chlorides, inorganic ororganic buffers, such as alkali metal phosphate buffer, amino acids,such as aspartic acid or glutamic acid and the salts thereof, organiccarboxylic acids, such as citric acid, organic nonvolatile solvents,such as DMSO, and other compounds, such as β-carotene and mixtures ofthese.

The microorganism cultures according to the invention preferablycomprise viable microorganisms in a concentration of 10⁸ to 10¹² cfu(colony forming units)/g of dry culture. The powder concentratesproduced according to the invention comprise from about 5·10⁸ to 1·10¹²,preferably about 4·10¹¹ to 8·10¹¹ cfu/g. The compressed culturesaccording to the invention comprise from about 1·10¹¹ to 4·10¹¹, inparticular about 3·10¹¹ cfu/g. Starter cultures for producing silagecomprise from about 1 to 7·10¹⁰, in particular about 3·10¹⁰ cfu/g.

In this process the microorganisms can be derived from one microorganismspecies or a plurality. A particularly preferred species arelactic-acid-producing bacteria, such as those which are suitable forsilage production, such as, for example, Lactobacillus plantarum.

For the purposes of the invention, silage comprises feed plant productswhich have been preserved by the action of microorganisms, for examplethose based on grass, clover, straw, corn plants, fodder beets, legumes,cereals, such as corn and wheat, and the like.

The second object of the present invention described above issurprisingly achieved by providing a spray-drying process for producinga dry microorganism culture, comprising at least one microorganismspecies in carrier-bound form, which comprises

-   -   a) dissolving or suspending at least one substance suitable for        forming a carrier in a liquid comprising at least one        microorganism species,    -   b) drying the resultant mixture in a spray-dryer, for the        spray-drying use being made of a conditioned dried gas heated to        a temperature in the range of above about 80° C., in particular        from about 90 to about 135° C., preferably from about 100 to        about 110° C., such as about 105° C., and    -   c) removing the dried material from the spray-dryer, this dried        material having an exit temperature of from about 40 to 85° C.,        in particular from about 45 to 75° C., preferably from about 50        to 65° C., such as about 55° C.

This spray-drying process according to the invention is also calledcarrier-bound spray-drying process below. The gas used for the drying ispreferably a dried gas having a dew point of below +5° C. in particularhaving a dew point of from about −10 to about −50° C., such asconditioned compressed air or conditioned nitrogen. For example,compressed air having a dew point of about −25° C. and nitrogen having adew point of about −40° C. can be used. A dew point of +5° C. isequivalent to roughly 5 g of water per m³ of air.

According to a preferred embodiment of the spray-drying processaccording to the invention, in a downstream further stage d), the driedmaterial is subjected to a post-drying. The post-drying temperature isin the range of from about 15 to 50° C., such as from about 25 to 40° C.The post-drying is performed, for example, in a gas atmosphere or invacuo; alternatively to this, there is also the possibility of mixing adesiccant homogeneously with the dry microorganism preparation obtainedin accordance with stage c).

Because of its design, the spray-drying process according to theinvention surprisingly permits microorganism suspensions to be dried atsurvival rates of up to 100%. Owing to the use of conditioned gas in thespray-drying as well as the optional post-drying step, surprisingly, drypreparations having an extremely low moisture content (of from about 2to 3% by weight of water), corresponding to a water activity a_(w) offrom 0.03 to 0.15, are provided. This directly causes the microorganismcultures which have been spray-dried according to the invention, with orwithout post-drying, to have survival rates of up to 60% after storagefor 1 year at ambient temperature and ambient air conditions.

Owing to the surprisingly high survival rate in the above-describedspray-drying, the content of viable microorganisms is markedly high. Theresultant spray-dried product is therefore also called powderconcentrate and, to reduce the concentration of viable cells, can befurther diluted, depending on the field of application. The powderconcentrate is particularly suitable for preparing the above-describedcompressed particulate cultures according to the invention.

The present invention therefore also relates to a process for producingthe above-described compressed microorganism cultures, which comprises

-   -   i) producing a powder concentrate of the microorganism culture        by carrier-bound spray-drying, carrier-bound freeze-drying or        carrier-bound fluidized-bed drying,    -   ii) with or without admixing the powder concentrate with one or        more coformulants and    -   iii) compressing this mixture by compacting or tabletting.

Preferably, in a further process step, the compressed mixture is broken,i.e. comminuted, and may be classified to give compressed granules ofthe desired size using a screen of suitable mesh width.

The present invention further relates to a process for producing a dryagglomerated microorganism culture, which comprises

-   -   i) producing a powder concentrate of the microorganism culture        by carrier-bound spray-drying, carrier-bound freeze-drying or        carrier-bound fluidized-bed drying,    -   ii) with or without admixing the powder concentrate with one or        more coformulants and    -   iii) compressing this mixture by agglomeration.

Carrier-bound means here the presence of at least one matrix material ofthe above-described type during drying.

According to a preferred embodiment of the above-described compactingprocess or tabletting process or agglomeration process, stage i) iscarried out in particular in accordance with the above-describedspray-drying process.

The product obtained by the above-described compression processes is,for the purposes of the present invention, also called compressed orcompacted dry concentrate (in the cfu range from about 1·10¹⁰ to 1·10¹¹)and can be marketed as such, e.g. as a concentrated starter culture.

The present invention further relates to the use of the compressed drymicroorganism cultures according to the invention as starter culturesfor producing foodstuffs, such as for the production of milk products,such as sour cream, buttermilk, yogurt, kefir, coumis, curd cheese, forproducing sourdough, uncooked sausage, and for producing feedstuffs,such as silage. For this purpose, the culture, with or withoutdissolution, is mixed with the foodstuff substrate or feedstuffsubstrate. If the cell count in the starter culture should be too high,it may be diluted, e.g. by mixing with an inert solid, such as lime, inparticular feed lime.

The present invention further relates to foodstuffs and feedstuffs whichhave been produced using the starter cultures according to theinvention.

The present invention is described in more detail in the sections nowfollowing with reference to the accompanying figure.

FIG. 1 shows diagrammatically a possible way of producing, from powderconcentrate, granules compacted in accordance with the invention.

USABLE MICROORGANISMS

The present invention is not restricted in principle to certainmicroorganism cultures. Rather, those skilled in the art recognize thatthe present invention is applicable to any microorganisms, in particularbacteria and yeasts, which can be converted to a dry microorganismpreparation under the conditions specified in the present description. Asuitable group of microorganisms which can be used according to theinvention are the group of lactic-acid-producing bacteria. Inparticular, these are bacteria which are suitable for thehomofermentative lactic acid fermentation, i.e. break down glucose tolactate via the fructose bisphosphate pathway. Typical representativesof this group are bacteria of the genera Lactobacillus sp.,Streptococcus sp. and Pediococcus sp. Concrete examples of lactobacilliwhich may be mentioned are Lactobacillus bulgaricus, Lactobacillusacidophilus, Lactobacillus helveticus, Lactobacillus bifidus,Lactobacillus casei, Lactobacillus lactis, Lactobacillus delbrueckii,Lactobacillus thermophilus, Lactobacillus fermentum, Lactobacillusbrevis and Lactobacillus plantarum. Examples of suitable streptococciare Streptococcus lactis, Streptococcus cremoris, Streptococcusdiacetilactis, Streptococcus thermophilus, Streptococcus pyrogenes,Streptococcus salivarius, Streptococcus faecalis, Streptococcus faecium;and examples of suitable pediococci are Pediococcus cerevisiae andPediococcus acidilactici.

Fermentation of the Microorganisms

To carry out the present invention, preferably, use is made of freshlyprepared microorganism suspensions. The fermentation media orfermentation conditions optimum for each microorganism are either knownfrom the prior art or can be determined in only a few routineexperiments by the person skilled in the art who is entrusted with theculture of microorganisms.

However, usually, the fermentation is carried out in such a manner thatstarting from a liquid or semi-solid preliminary culture (culture volumefrom about 10 to 200 ml), freshly prepared sterile fermentation mediumis inoculated under sterile conditions, where the volumetric ratio ofpreliminary culture to culture medium can be from about 1:50 to 1:200.Preferably, freshly grown preliminary cultures are used which are in alate phase of logarithmic growth. Depending on the microorganism beinggrown, they are cultured under specific optimized growth conditions(such as temperature and pH). Usually, the growth temperature is in therange from about 20 to 40° C., but, for example when thermophilicbacteria are being grown, markedly higher temperatures can be present.The fermentation batch is kept uniformly agitated, for example bymoderate stirring or introducing air or nitrogen in order to prevent thedevelopment of temperature or substance gradients and to ensurecontinuous growth in this manner. After the growth phase is complete(determined for example by achieving a defined cell density orconsumption of one of the added nutrients), the cell suspension can beused directly to produce the dry preparations according to theinvention.

However, it is also possible to concentrate the resultant original cellsuspension to increase the cell count. Suitable methods for this are,for example, centrifugation, ultrafiltration or thin-film evaporation.However, a centrifugation step is usually used to concentrate the cellsuspension, which centrifugation step is preferably carried out at adecreased temperature, that is to say in the range from about 4 to 10°C.

Instead of the concentration, or in combination with it, there is alsothe possibility of subjecting the freshly cultured cell suspension to awashing step in order to remove culture constituents, such as metabolicproducts, which may have an adverse effect on the activity. In thiscase, the procedure usually adopted is that, preferably at from about 4to 10° C., the original culture broth is first concentrated to give asuspension of high cell density and this is then taken up in a suitablebuffer solution and diluted to the desired cell density. If necessary,the washing step can also be repeated a plurality of times. Solidscontents which can be used according to the invention of cultures ofmicroorganisms suitable for producing the dry preparations according tothe invention are usually in the range from about 5 to 25% by weight,such as from about 10 to 20% by weight.

The microorganisms can be cultured by batch fermentation orcontinuously.

To further illustrate the invention, in the section below, a moredetailed description is given of culturing a lactic acid bacterium, inparticular Lactobacillus plantarum. This is a bacterium which is to befound in particular on intact and decomposing plants and is particularlysuitable for producing silage feedstuffs.

A suitable fermentation medium comprises, per liter of medium, fromabout 40 to 60 g of glucose, from about 30 to 60 g of yeast extract anda cocktail of customary trace elements, such as magnesium, manganeseand, optionally, iron. The pH of the fermentation medium is from about 6to 7. The fermentation temperature is from about 33 to 38° C. The pH ofthe fermentation medium can be kept within the desired range by addingsterile sodium hydroxide solution. Growth is complete when glucoseconsumption or lactic acid synthesis can no longer be observed.

According to a lactobacillus fermentation variant which is suitableaccording to the invention, after about 80% or 90% of the growth isachieved, the fermentation medium temperature is increased to from about42 to 46° C. until the added glucose is completely consumed. It has beenfound according to the invention that cultures produced in this mannerare particularly stable in particular in the spray-drying, as a resultof which high survival rates are achievable. Comparable growth variantsare also conceivable with other microorganisms which can be usedaccording to the invention.

After growth is complete, the fermentation batch is brought to thedesired cell density. If desired, the cell suspension can be washeduntil it is virtually lactate free. The cell count of a microorganismsuspension suitable according to the invention is usually in the rangefrom about 1×10¹⁰ to about 5×10¹² cfu/g of suspension.

Carrier Substances

The dry microorganism cultures prepared according to the invention, inaddition to any nonvolatile constituents present from the respectivefermentation batch, such as metabolic products, comprise at least onematrix material with or without other stabilizing substances. Thesecoformulants are preferably selected from inorganic salts or buffers, atleast one other compound which is selected from mono-, oligo- andpolysaccharides, polyols, polyethers, amino acids, oligo- andpolypeptides, milk-derived compounds, organic carboxylic acids, mineralcompounds, organic carrier materials such as wheat semolina bran,alginates, DMSO, PVP (polyvinylpyrrolidone), CMC(carboxymethylcellulose), α-tocopherol, β-carotene and mixtures thereof.

Examples of suitable saccharide carrier components are sucrose,fructose, maltose, dextrose, lactose and maltodextrin. An example of asuitable polyol is glycerol. Examples of suitable amino acids areglutamic acid, aspartic acid and the salts thereof. An example of asuitable peptide carrier is peptone. An example of a milk-derivedcompound is, in addition to the abovementioned maltodextrin, also sweetwhey powder. Suitable organic carboxylic acids are, for example, citricacid, malic acid and L-ascorbic acid. Examples of suitable mineralcarriers are montmorillonite and palygorskite.

However, preferably, as carrier for the dry microorganism preparationsaccording to the invention, use is made of mixtures of theabovementioned classes of substances. Mixtures of this type preferablycomprise, as main component, a matrix material, such as one of theabovementioned saccharide components or, for example, sweet whey powder,with or without a minor content of at least one further component, suchas a buffer component (for example citric acid) or an antioxidant (forexample L-ascorbic acid or α-tocopherol). The addition of furtherstabilizing constituents, such as sodium glutamate and/or peptone, haslikewise proved to be advantageous.

The matrix component is customarily used in carrier compositions usableaccording to the invention in about 5 to 30 times the amount of theother carrier constituents. Examples of particularly suitable carriercombinations are:

-   -   a) sweet whey powder/citric acid/L-ascorbic acid (weight ratio        about 40:1:1).    -   b) maltodextrin/lactose/citric acid/L-ascorbic acid (weight        ratio about 20:20:1:1), unsupplemented or supplemented by about        1.5 parts of β-carotene and 0.5 part of α-tocopherol per part of        citric acid.    -   c) maltodextrin/sodium glutamate/L-ascorbic acid (weight ratio        about 10:1.5:1).    -   d) lactose/glucose/peptone/citric acid (weight ratio about        6:6:1.2:1).

The carrier substances according to the invention can be added to themicroorganism suspension either as solid or in dissolved form. However,preferably, a sterile solution of the carrier/carriers is prepared, thisis cooled to a temperature of from 4 to 10° C. and this is mixed withthe likewise cooled microorganism suspension with gentle stirring. Toprepare a homogeneous suspension, the resultant mixture is stirred withfurther cooling for a period of from about 10 minutes to 1 hour.

Preparation of Dry Microorganism Preparations

The microorganism suspension containing the carrier added in the mannerdescribed above can then be dried in various ways. Suitable dryingprocesses are in principle freeze drying, fluidized-bed drying and,preferably, spray-drying. For the purposes of the present invention,spray-drying also comprises modified spray-drying processes, such asspray-agglomeration or agglomerating spray-drying. The latter process isalso known under the name FSD (fluidized spray-dryer) process.

Freeze-drying for preparing dry microorganism cultures according to theinvention can be carried out, for example, on the basis of thefreeze-drying process described in EP-A-0 259 739 or U.S. Pat. No.3,897,307. The contents of these publications are hereby incorporatedcompletely by reference.

A suitable fluidized-bed drying process is described, for example, inthe dissertation by U. Kessler on the subject “ExperimentelleUntersuchung und Modellierung der Überlebensrate von Milchsäurebakterienbei der thermischen Trocknung” [Experimental study and modeling of thesurvival rate of lactic acid bacteria during thermal drying], TechnicalUniversity of Munich, 1993. The contents of this publication arelikewise incorporated completely by reference. To carry out thefluidized-bed drying process, it is advantageous that the carriermaterial to be used, in particular the matrix component, is introducedin a fluidized bed and this is sprayed with the microorganism suspensionin the manner described by U. Kessler.

However, the drying process which is most preferred according to theinvention is spray-drying. Those methods which can be used according tothe invention are essentially all spray-drying techniques knownhitherto. The material to be sprayed can, for example, be driedcocurrently or countercurrently; spraying can be carried out by means ofa single-component or multiple-component nozzle or by means of anatomizer wheel.

Preference is given according to the invention to the use of material tobe sprayed having a solids content (after addition of the carrier) offrom about 10 to 40, such as from about 10 to 25% by weight.

The spray-drying process according to the invention is carried out insuch a manner that a conditioned dry gas having a temperature in therange of above about 80° C. is introduced into the drying apparatus. Inparticular, the inlet temperature should be in the range of from about90 to 135° C. Particular preference is given to a drying temperature inthe range of about 105° C. The rate of the drying process is designedaccording to the invention in such a manner that the exit temperature ofthe drying material from the dryer is in the range of about 45 to 75°C., in particular in the range of from about 50 to 65° C., preferablyabout 55° C.

Of particular importance to the process according to the invention isthe use of preconditioned, i.e. low-moisture, drying air. Preferably,use is made of compressed air having a dew point at about −25° C.

The drying process according to the invention shall be carried out insuch a manner that a very low residual moisture content is present inthe dry material. Preferably, the water activity a_(w) in the dryingmaterial should be less than 0.4. However, to further improve thelong-term storage stability, according to the invention water activitiesof less than 0.15, preferably in the range from about 0.03 to 0.1 aresought after. The percentage water content is preferably from about 2 to3% by weight. Most preferably, this is achieved by adding a post-dryingstep subsequently to the spray-drying step. The drying material for thispurpose is, for example, post-dried in a fluidized bed, preferably at atemperature in the range of from 15 to 50° C., for a period of, forexample, from 15 minutes to 20 hours. Again, preferably, conditionedcompressed air or conditioned nitrogen serves as drying gas. However,the post-drying can also be performed by applying a vacuum of from about1 to 50 mm Hg for a period of from about 15 minutes to 20 hours and at atemperature of from about 15 to 50° C. In this case, preference is givento stirring the drying material, for example, using a paddle agitator.

Instead of the above-described physical post-drying processes, it isalso conceivable to add specific desiccants to the dry material obtainedfrom the spray-drying. Desiccants of this type should themselves have avery low water activity, such as an a_(w) of 0.01 or less. Examples ofsuitable desiccants are inorganic salts, such as calcium chloride andsodium carbonate, organic polymers, such as the product obtainable underthe trade name Kollidion 90 F, and silicon-dioxide-containingdesiccants, such as silica gel, zeolites and desiccants which areobtainable under the trade name Tixosil 38, Sipernat 22 S or Aerosil200.

According to the invention, it was surprisingly found that, despite therelatively high drying temperatures, the survival rate for the drypreparations according to the invention had excellent values, namely of75%±25%.

The content of viable microorganisms is in the range of from about 5×10⁸to 1×10¹² cfu/g of dry matter. These preparations are also calledaccording to the invention powder concentrates. Since, for individualfinal applications, lower contents of viable microorganisms are alsocompletely sufficient, powder concentrates of this type can therefore ifappropriate be blended to the final count of viable microorganisms bymixing with further inert carrier material.

Preparation of Compressed Dry Microorganism Cultures

The powder concentrates obtainable by the above-described dryingprocesses usually have a relatively high dust content and are thus notyet satisfactorily handleable for individual applications. Furthermore,various applications require an increased mechanical stability of thedry cultures. It is therefore necessary to improve the properties of theabove-described powder concentrates by a further compression step.

To reduce the dust content of the powder concentrates according to theinvention, it is possible to agglomerate these in a conventional mannerto form granules, or using external forces, to compact them or tabletthem.

Agglomeration is a generally known process, and is described, forexample, by Schade, A. and Leuenberger, H. in Continuous fluidized-bedspray granulation, Chemie Ingenieur Technik (1992) 64 (1992) 1016;Uhlemann, H., Preparation of pharmaceutical granules in a combined wetgranulation and multichamber fluidized-bed drying process, ChemieIngenieur Technik 62 (1990), 822; or Rosch, M. and Probst R.,Granulation in the fluidized bed, Verfahrenstechnik (1975), 9, 59.

Use can be made according to the invention of agglomeration using amixer. For this purpose, the above-described powder concentrate ischarged into the mixer and oil, water or an aqueous or alcoholicsolution of sugars, polymers or other additives is sprayed in toagglomerate the powder concentrate.

In addition, use can be made according to the invention of agglomerationin a fluidized bed. In this case, powder concentrate is vortexed withgas feed and sprayed with an aqueous or alcoholic solution of sugars,polymers or other additives to form the agglomerate. Suitable processesfor this purpose are described, for example, in WO-A-88/06181, in thedissertation by U. Kessler (loc. cit.) and by K. Fuchs in ZFL 45 (1994)31. The disclosure of the abovementioned publications is herebyincorporated by reference.

Agglomeration produces granulated microorganism cultures having aparticle size in the range of from about 0.1 to about 4 mm, inparticular from about 0.3 to 2.5 mm.

However, particularly preferred according to the invention is thepreparation of dry microorganism cultures which are present in the formof particularly highly compressed particles. This is carried outaccording to the invention either by tabletting in conventional tabletpresses or with the use of conventional compacting apparatuses equippedwith two counter-rotating rolls.

To compress the powder concentrates obtainable according to theinvention, to these are usually added one or more coformulants oradditives to modify the processability to the end product or theproperties of the end product.

To improve the flowability of the powder concentrate, a free-flowingagent is preferably added. Examples of a suitable free-flowing agent arespray-dried silicon dioxide powders, which are obtainable, for example,under the trade name Sipernat 50. To improve the storage stability ofthe solid formulations according to the invention, in addition,conventional antioxidants, such as L-ascorbic acid, can be added.Furthermore, desiccants of the above-described type can additionally beadded.

The action of the cultures according to the invention is markedlyimproved if measures are taken which, after the culture has beenapplied, lead to a rapid breakdown of the grain structure and thus to arapid release of the microorganisms. One possibility of achieving thisis the addition of a readily water-soluble component which thusaccelerates the breakdown of the grain structure. Suitable compoundsare, for example, poly(ethylene glycol)s, which are obtainable, forexample, under the trade name Pluriol E.

Another solid formulation particularly preferred according to theinvention comprises what is termed an effervescence additive. This is agas-releasing component, in particular a CO₂ source, for example analkaline earth metal hydrogen carbonate, preferably sodium hydrogencarbonate or ammonium hydrogen carbonate; and an acid component,preferably selected from citric acid, ascorbic acid or malic acid. Thiseffervescence additive, in the presence of moisture, produces aspontaneous gas formation with breakdown of the grain structure andrapid release of the microbial cells.

In particular, to prepare highly compressed, compacted or tablettedmicroorganism cultures, it is advisable to add compacting or tablettingaids. This is because it has surprisingly been found according to theinvention that adding such compacting aids decreases the pressuresacting on the microorganisms during the compacting and thus markedlyimproves the survival rate of the microbes. Examples of suitablecompacting aids are microcrystalline cellulose, sugars and mixturesthereof. Concrete examples of microcrystalline cellulose are productswhich are commercially available under the trade names Avicel, Arboceland Vivapur. Examples of suitable sugars are maltose, maltodextrin andlactose preparations, which are obtainable under the trade namesGranulac, Tablettose or FloLac. An example of a suitable mixedcellulose/sugar product is the commercial preparation Cellactose. Afurther suitable tabletting aid is a lactose preparation granulatedusing PVP, obtainable under the trade name Ludipreβ.

Other suitable additives are poly(ethylene glycol)s (Mw from 100 to10,000) which can have a stabilizing action on the cells embedded in thematrix.

The accompanying FIG. 1 shows a flow diagram for the further processingaccording to the invention of the powder concentrates to give acompacted product according to the invention. Powder concentrate PK ismixed in the mixer M1 with the coformulants or additives ZU, passes fromthere into a reservoir B1 which feeds the compactor A1. The productribbon exiting from the compactor is precomminuted or finely comminutedin the grinders Z1 and Z2 and in the screen F1, product PR is separatedoff from dust fractions having a particle size of less than 0.3 mm. Thismaterial is fed to the mixer M1 as recycled material RÜ. The product PRhaving a particle size of 0.3 mm or above, such as from 0.3 to 1.5 mm,passes to the packaging station or may be subjected to furtherprocessing, such as a coating process.

Suitable coating materials, which preferably are additionally to hinderthe ingress of moisture to the dry preparation, are, for example,alcoholic solutions of PVP, in particular a PVP product which iscommercially available under the trade name Kollidon VA64. Anotherusable coating system is a mixture of shellac and Kollidon 25 or 30,which is supplemented with titanium dioxide and tallow and is likewisepresent in alcoholic solution.

To reduce the cell count further if necessary, a coated or uncoatedproduct obtained in this manner can be blended, for example, with lime,or another suitable mineral additive.

EXAMPLES Analytical Methods Used in the Following Examples

a) Cell Count Determination:

Cell counts are determined in the conventional manner by serial dilutionwith sterile 0.9% strength NaCl solution and subsequent plating on MRSagar (Difco Laboratories). Colony-forming units (cfu) were counted afterincubation for 48 hours at 37° C. Only plates which contained between 30and at most 300 colonies were evaluated. Generally, 3 plates per stagewere evaluated and the mean taken.

The specific cell count of a sample was determined by calculation,dividing the cell count per gram of sample by the relative sample drymatter content.

b) Determination of the Survival Rate on Drying:

The survival rate during drying was calculated from the specific cellcount of the sample before drying divided by the specific cell countafter drying. It was always expressed in percent.

c) Determination of Storage Stability:

To determine the storage stability of a dried sample, the specific cellcount of the dried sample was determined immediately after drying(day_(O)). The dried cell material was stored under air in an opaquetightly sealed vessel at room temperature (21° C.±2° C.) for extendedperiods. The specific cell count was determined again at regularintervals (day_(N)). The storage stability was calculated from thequotient of specific cell count day_(N)/specific cell count day_(O).

If the specific cell count after drying was, for example, 5·10¹¹ cfu/gof DM and, after storage for 8 weeks, 4·10¹¹ cfu/g of DM, the storagestability was 80% of the initial value.

d) Moisture Content Measurement:

Electronic moisture analyzer HR 73 from Mettler Procedure: approximately2 g of powder are distributed onto the measuring scales of theinstrument. Measurements are taken at a drying temperature of 105° C. upto constant weight (switch-off criterion: max. 1 mg of weight loss in 50seconds).

e) Measurement of Water Activity:

Hygroscope DT instrument from Rotronic AG, Zürich, Switzerland Theproduct is placed in the sample holder and this is positioned in themeasuring chamber thermostatted to 25° C. After closing the measuringchamber and an equilibration time of 20 minutes, the instrumentmeasurement value is read off.

f) DSC Measurement to Determine the Glass Transition Temperature T_(g):

TA4000 instrument from Mettler Sample weight approximately 15 mg,heating rate 20° C./min, samples were flushed with a nitrogen streamduring measurement.

-   -   DSC=Differential Scanning Calorimetry

Microorganism Culture Examples Example K1 Batch Fermentation 10 LiterScale

10 l of a fermentation medium which comprised the following constituentswere placed in a 14 1 fermenter and sterilized at 121° C. for 30minutes:

Glucose monohydrate 550.0 g 50% yeast extract suspension 750.0 g (pH 4.5with phosphoric acid) Tween 80 ® 10.0 g MgSO₄ * 7 H₂O 5.0 g MnSO₄ * 1H₂O 0.5 g

After sterilization, the medium was adjusted to pH 5.8 at 37° C. usingsterile 25% strength sodium hydroxide solution and the medium wasblanketed with a gentle stream of sterile nitrogen. The fermenter wasstirred at 150 rpm.

The fermenter was then inoculated with 100 ml of a preculture ofLactobacillus plantarum (BASF strain LU 3244) which had previously beengrown for 16 h at 37° C. in MRS nutrient medium (Difco Laboratories).The culture pH was continuously kept at 6.2 using 25% strength sodiumhydroxide solution.

The course of the fermentation was followed from the sodium hydroxidesolution consumption. As soon as no more sodium hydroxide solution wasconsumed (total consumption 890 g), all of the fermentation broth wasdrained off and centrifuged at 8° C. The biomass was resuspended inabout 600 g of supernatant and made up to exactly 1000 g withsupernatant. The dry matter content was determined using an infrareddrying balance (105° C. to constant weight). The solids content of thissuspension was 15%.

Example K2

Batch Fermentation 200 Liter Scale

180 l of a fermentation medium which comprised the followingconstituents were placed in a 200 1 fermenter and sterilized at 121° C.for 30 minutes:

Glucose monohydrate 11 kg 50% yeast extract suspension 15 kg Tween 80 ®200 g MgSO₄ * 7 H₂O 200 g MnSO₄ * 1 H₂O 10 g

After sterilization, the medium was adjusted to pH 5.8 at 37° C. usingsterile 25% strength sodium hydroxide solution and the medium wasblanketed with a gentle stream of sterile nitrogen.

The fermenter was then inoculated with 2000 ml of a preculture ofLactobacillus plantarum (3244) which had previously been grown for 24 hat 30° C. in MRS nutrient medium. The pH of the culture was continuouslycontrolled using 25% strength sodium hydroxide solution.

The course of the fermentation was followed from the sodium hydroxidesolution consumption. In total, 16.43 kg of 25% strength NaOH wereconsumed. As soon as sodium hydroxide solution was no longer consumed,all of the fermentation broth was drained off and harvested at 8° C.using a continuous separator. The harvested biomass had a weight aftercentrifugation of 20 kg, and the solids content of this suspension was12.3%. The cell count of the suspension was 1.04·10¹¹ cfu/g ofsuspension. The specific cell count was 8.45·10¹¹ cfu/g of dry matter(DM).

Example K3 Batch Fermentation With Temperature Shock

A fermentation was carried out in a similar manner to Example 2. At asodium hydroxide consumption corresponding to 90% of the expected value,the fermenter temperature was increased to 44° C. and kept until all ofthe sugar present had been consumed. The cells were then harvested asdescribed in Example K2. The cell count of the fermentation broth was1.8·10¹¹ cfu/g at a solids content of 21.17%. This corresponds to aspecific cell count of 8.5·10¹¹ cfu/g DM.

Example K4 Continuous Fermentation

10 l of a fermentation medium having the following composition werecharged into a 14 l fermenter and sterilized at 121° C. for 30 minutes(production fermenter):

Glucose monohydrate 400.0 g 50% yeast extract suspension 500.0 g (pH 4.5with phosphoric acid) KH₂PO₄ 30.0 g Citric acid monohydrate 21.0 g Tween80 ® 10.0 g MgSO₄*7 H₂O 5.0 g MnSO₄*1 H₂O 1.7 g (NH₄)₂Fe(SO₄)₂* 6H₂O 0.4g

2000 l of the same medium were charged into a second fermenter having atotal volume of 3000 and sterilized (reservoir fermenter). Bothfermenters were connected by a sterilizable line. Via an intermediatevessel which stood on a balance, using an automatic control system, 3 lof fresh medium were pumped every hour into the production fermenter.The temperature of the production fermenter was controlled to 37° C. ThepH was controlled to 5.8 using 25% strength NaOH. The fermenter wasstirred at 150 rpm and blanketed with nitrogen at 0.1 VVM.

Via a second pump, 3 l of medium were continuously taken off every hourand collected in a stainless steel collection vessel precooled to from 0to 4° C. The biomass concentration was determined by turbidimetry andwas 3.5 g/l. The glucose, concentration in the production fermentereffluent was, after the initial growth phase, 0 g/l at all times. Thecell count of the fermentation broth was 1.48·10¹⁰ cfu/g of broth. Thedry matter content of the fermentation broth was 6.89%, equivalent to217 g DM. The specific cell count was 2.15·10¹¹ cfu/g of DM.

Example K5 Cell Harvest With Washing Step to Remove Sodium Lactate

72 l of fermenter discharge from Example K4 were harvested continuouslyat 8° C. using a commercial separator. About 7 kg of cell suspension wasobtained. To this was added a washing solution which comprised 40 l ofdeionized water, 450 g of NaCl and 136 g of KH₂PO₄. The pH of thewashing solution had been adjusted in advance to 7.0 using 25% strengthsodium hydroxide solution. The about 50 l of resuspended cells wereagain separated. 3160 g of concentrated washed cell suspension wereobtained. The solids content of the suspension was 9.97%. The cell countwas 2.49·10¹¹ cfu/g of suspension. The specific cell count was 2.5·10¹²cfu/g DM.

This washed cell suspension was virtually free of sodium lactate. Thebiomass concentration was determined by turbidimetry to be 80 g/l.

Examples of Preparation by Spray-Drying of Powder Concentrates Accordingto the Invention

The spray-drying experiments described in the following section forpreparing powder concentrates according to the invention are carried outin a laboratory spray-dryer of type Niro Minor from Niro, Copenhagen,Denmark. The ready-to-spray bacterial suspension is sprayed via atwo-component nozzle cocurrently with preconditioned heated compressedair into the plant drying tower, the dried product is separated from theair using a cyclone and collected.

Example S1

To prepare a coformulant solution, 200 ml of deionized water (completelydemineralized water) are heated to 60° C. 150 g of sweet whey powder,7.5 of NaCl, 3.8 g of KH₂PO₄, 3.8 g of citric acid and 3.8 g ofL-ascorbic acid are dissolved therein, adjusted to pH 7 using 40%strength aqueous NaOH and made up to 400 g total mass using deionizedwater. This solution is cooled to 5° C.

200 ml of washed, i.e. essentially sodium-lactate-free centrifugedferment (prepared in a similar manner to Example K5) (12.7% solidscontent (S.C.)) are placed in an ice bath at a temperature of 5° C. and400 g of coformulant solution, cooled to 5° C., are added with stirring.The mixture of centrifuged ferment and coformulants is further stirredfor 30 minutes at 500 rpm using a magnetic stirrer with ice bathcooling. By means of spray-drying (Niro Minor apparatus) the mixture isthen converted into a powder concentrate A, which is separated off inthe cyclone. In the course of this, the reservoir from which the mixtureis metered is cooled to 4° C., the inlet temperature is from 105 to 110°C., the exit temperature is from 53.5 to 55.5° C. A two-component nozzleis used, conditioned air (dew point −25° C.) at 4 bar being used tospray the mixture of centrifuged ferment and coformulants.

The powder concentrate A is further dried at room temperature for 2hours in a nitrogen-operated (dew point=−40° C.) fluidized bed, powderconcentrate B being obtained.

Characterizations:

-   -   Ready-to-spray mixture: 35% S.C., 2.84−10¹¹ cfu/g of dry matter    -   Powder concentrate A: water activity a_(w)=0.135    -   Powder concentrate B: water activity a_(w)=0.076,        -   moisture content 3.4%,        -   T_(g) from DSC measurement: 54° C.,        -   1.98·10¹¹ cfu/g of dry matter (equivalent to 70% survival            rate in the drying)    -   Storage study of powder concentrate B: cfu counts with        room-temperature storage in containers sealed under ambient air:        2.0·10¹¹ cfu/g of dry matter (100%) after 30 days

Example S2

To prepare a coformulant solution, 200 ml of deionized water are heatedto 70° C. 75 g of maltodextrin (Glucidex IT6, Roquette), 75 g oflactose, 7.5 g of NaCl, 3.8 g of KH₂PO₄, 3.8 g of citric acid and 3.8 gof L-ascorbic acid are dissolved therein, the mixture is adjusted to pH7 using 40% strength aqueous NaOH and made up to 400 g total mass usingdeionized water. This solution is cooled to 5° C.

200 ml of washed, i.e. essentially sodium-lactate-free centrifugedferment (16.5% S.C.; prepared similarly to example K5) are mixed, at 5°C., with stirring into 400 g of coformulant solution, cooled to 5° C.The mixture is stirred for 30 minutes at 250 rpm by a magnetic stirrerwith ice bath cooling. 101 ml of a solubilized mixture prepared inaccordance with EP-A-0 479 066 (BASF) from 25% Tween 80, 5% β-caroteneand 2% α-tocopherol are then added and further stirred for 10 minuteswith ice bath cooling. This mixture is then converted by spray-drying,as described in Example S1, into a powder concentrate A (inlettemperature 105° C., exit temperature from 54 to 55° C.). The powderconcentrate A is not further dried.

Characterizations:

-   -   Ready-to-spray mixture: 29% S.C., 3.84·10¹¹ cfu/g of dry matter    -   Powder concentrate A: water activity a_(w)=0.065,        -   moisture content 2.8%,        -   T_(g) from DSC measurement: 61° C.,        -   2.22·10¹¹ cfu/g of dry matter (equivalent to 58% survival            rate in the drying)            Storage Study on Powder    -   concentrate A: cfu counts for room-temperature storage in        containers sealed under ambient air:        -   1.9·10¹¹ cfu/g of dry matter (86%) after 30 days

Example S3

400 ml of unwashed, i.e. sodium-lactate-containing, centrifuged ferment(prepared similarly to Example K4)(14.3% S.C.) are placed in an ice bathat a temperature of 5° C. 57.2 g of Glucidex IT6, 8.6 g of L-ascorbicacid and 5.7% of sodium glutamate are stirred as solids into the cooledcentrifuged ferment with stirring at 700 rpm by means of a magneticstirrer. The pH is adjusted to 7 using 40% strength aqueous NaOH. Themixture of centrifuged ferment and coformulants is further stirred for30 minutes at 500 rpm using a magnetic stirrer at approximately 3° C.with ice bath cooling. The mixture is then converted by spray-drying, asdescribed in Example S1, into a powder concentrate A (inlet temperature105° C.; exit temperature from 54.5 to 55.5° C.).

The powder concentrate A is further dried at room temperature in anitrogen-operated fluidized bed for 2 hours, a powder concentrate Bbeing obtained.

Characterizations:

-   -   Ready-to-spray mixture: 27% S.C., 4.65·10¹¹ cfu/g of dry matter    -   Powder concentrate A: water activity a_(w)=0.197    -   Powder concentrate B: water activity a_(w)=0.072,        -   moisture content 3.8%,        -   T_(g) from DSC measurement: 52° C.,        -   4.64·10¹¹ cfu/g of dry matter (equivalent to 100% survival            rate in the drying)            Storage Study on Powder Concentrate B:    -   cfu counts with room-temperature storage in containers sealed        under ambient air:        -   45 4.1·10¹¹ cfu/g of dry matter (88%) after 28 days

Example S4

215 ml of washed, i.e. essentially sodium-lactate-free, centrifugedferment (prepared similarly to Example K5) (14.5% S.C.) are placed in anice bath at a temperature of 5° C. 31.2 g of Glucidex IT6, 4.7 g ofascorbic acid and 3.1% of sodium glutamate are then stirred in as solidsinto the cooled centrifuged ferment with stirring at 700 rpm by amagnetic stirrer. The pH is adjusted to 7 using 40% strength aqueousNaOH. The mixture of centrifuged ferment and coformulants is furtherstirred for 30 minutes at 500 rpm using a magnetic stirrer with ice bathcooling. The mixture is then converted by spray-drying, as described inExample S1, into a powder concentrate A (inlet temperature 105° C.; exittemperature from 54.5 to 55.5° C.).

The powder concentrate is further dried at room temperature in anitrogen-operated fluidized bed for 2 hours, powder concentrate B beingobtained.

Characterizations:

-   -   Ready-to-spray mixture: 28% S.C., 8.76·10¹¹ cfu/g of dry matter    -   Powder concentrate B: water activity a_(w)=0.044,        -   moisture content 3.8%,        -   T_(g) from DSC measurement: 48° C.,        -   7.17·10¹¹ cfu/g of dry matter (equivalent to 82% survival            rate in the drying)            Storage Study on Powder    -   concentrate B: cfu counts for room-temperature storage in        containers sealed under ambient air:        -   3.7·10¹¹ cfu/g of dry matter (52%) after 27 days

Example S5

To prepare a coformulant solution 1, 40 ml of deionized water arecharged and 33.3 g of lactose and 6.3 g of peptone are dissolvedtherein, the mixture is made up to a total mass of 83 g with deionizedwater and adjusted to pH 7 using 40% strength aqueous NaOH. To prepare acoformulant solution 2, 40 ml of deionized water are charged and 33.3 gof glucose-1-hydrate and 5.4 g of citric acid are dissolved therein, themixture is made up to a total mass of 83 g with deionized water andadjusted to pH 7 using 40% strength aqueous NaOH. These solutions 1 and2 are cooled to 5° C.

200 ml of washed, i.e. essentially sodium-lactate-free, centrifugedferment (prepared similarly to Example K5) (12.7% S.C.) are mixed with83 g of the cooled coformulant solution 1 in an ice bath atapproximately 4° C. The mixture is stirred for 30 minutes with ice bathcooling. 83 g of the cooled coformulant solution 2 are then added withstirring and further stirred for 30 minutes with ice bath cooling. Thenthis mixture is converted by spray-drying, as described in Example S1,into a powder concentrate A (inlet temperature 105° C.; exit temperature55° C.).

The powder concentrate A is further dried at room temperature for 2hours in a nitrogen-operated fluidized bed, powder concentrate B beingobtained.

Characterizations:

-   -   Ready-to-spray mixture: 29% S.C., 7.30·10¹¹ cfu/g of dry matter    -   Powder concentrate B: water activity a_(w)=0.139,        -   moisture content 3.7%,        -   T_(g) from DSC measurement: 45° C.,        -   5.06·10¹¹ cfu/g of dry matter (equivalent to 69% survival            rate in the drying)            Storage Study on Powder    -   concentrate B: cfu counts at room-temperature storage in        containers sealed under ambient air:        -   4.8·10¹¹ cfu/g of dry matter (95%) after 21 days

Example S6

The ready-to-spray mixtures were prepared in a similar manner to ExampleS3. Here, however, two different centrifuged ferments were used:

Example S6a: batch fermentation, with the ferment having been cooled to4° C. for 40 minutes toward the end of the fermentation.

The powder concentrate A obtained in the spray-drying in accordance withExample S1 (inlet temperature from 107 to 111° C.; exit temperature from58 to 61° C.) was not further dried.

Characterizations:

-   -   Ready-to-spray mixture: 3.68·10¹¹ cfu/g of dry matter    -   Powder concentrate A: 0.76·10¹¹ cfu/g of dry matter (equivalent        to 21% survival rate in the drying)

Example S6b: batch fermentation, with the ferment having been heated to44° C. toward the end of the fermentation. In this example, theready-to-spray mixture was divided. In a first experiment, the reservoirvessel was thermostatted to 4° C., as in Examples S1 to S5 and S6a. In asecond experiment, the reservoir vessel was thermostatted to 20° C.

The powder concentrates A obtained by spray drying in accordance withExample S1 (inlet temperature from 103 to 110° C.; exit temperature from59 to 61° C.) were not post-dried.

Characterizations for Rreservoir at 4° C.:

-   -   Ready-to-spray mixture: 3.53·10¹¹ cfu/g of dry matter    -   Powder concentrate A: 2.36·10¹¹ cfu/g of dry matter (equivalent        to 67% survival rate in the drying)        Characterizations for Reservoir at 20° C.:    -   Ready-to-spray mixtures:3.53·10¹¹ cfu/g of dry matter    -   Powder concentrate A: 1.48·10¹¹ cfu/g of dry matter (equivalent        to 42% survival rate in the drying)

Formulation Examples

In accordance with the formulas stated below, dry mixtures of powderconcentrates according to the invention were prepared and processed toform compacted starter culture preparations:

Unless specified otherwise, the release agent used was Leucine and thefree-flowing agent used was Sipernat 50S (spray-dried silicon dioxide).

The individual components of the preparations are first mixed with oneanother. For this purpose, for example, a plowshare mixer is used (typeLö 20 from Lödige). The dry mixture obtained in this manner is compactedin a compactor. For example, for this purpose a laboratory compactor canbe used which applies a pressing force of 14 kN/cm² (e.g. laboratorycompactor L 200 from Bepex). The product ribbon exiting from thecompactor is then comminuted to a particle size of≦1.25 mm. The crudegranules are screened to separate off fines of a particle size of≦0.3mm. The yield of useful material is about from 50 to 60% of the materialused.

Example F1 Preparing a Compacted Effervescent Product for Use as StarterCulture for Silage

Preparation A: Powder concentrate (in accordance with Example S2) 200.0g Citric acid, anhydrous 95.0 g NaHCO₃ 95.0 g PEG (M_(w) < 400) 8.0 gFree-flowing agent 2.0 g Preparation B: Powder concentrate (according toExample S2) 100.0 g Ascorbic acid, powder 47.5 g NaHCO₃ 47.5 g PEG(M_(w) < 400) 4.0 g Free-flowing agent 1.0 g Preparation C: Powderconcentrate (according to Example S2) 100.0 g Malic acid 47.5 g NaHCO₃47.5 g PEG (M_(w) < 400) 4.0 g Free-flowing agent 1.0 g Preparation D:Powder concentrate (in accordance with Example S2) 100.0 g Zeolite A(Wessalith P) 20.0 g Ascorbic acid, powder 37.0 g NaHCO₃ 36.8 g Releaseagent 3.0 g Free-flowing agent 3.0 g

Example F2 Preparation of a Quick-Dissolving Compacted Mixture WithoutEffervescent Additive

Powder concentrate (according to Example S2) 100.0 g water-solublesurfactant (Pluriol EL 500) 90.0 g Release agent 7.0 g Free-flowingagent 3.0 g

Example F3 Preparation of a Compacted Mixture

Powder concentrate (in accordance with Example S5) 100.0 g Compactingaid¹⁾ 90.0 g Release agent 7.0 g Free-flowing agent 3.0 g ¹⁾selectedfrom: Avicel PH 102, Vivapur 105, FlowLac, Maltex 20, Cellactose ormixtures thereof

Example F4 Preparation of Stabilized Compacted Mixtures

Base Formula:

Powder concentrate (according to Example S5) 100.0 g  Compacting aid50.0 g Stabilizer cf. Table I Release agent  7.0 g Free-flowing agent 3.0 g

TABLE I Stabilizer Component Amount (g) A Zeolite A 40 B PEG 4000 40 CAscorbic acid¹⁾ 40 D PEG 4000 20 Ascorbic acid 20 E Zeolite A 20Ascorbic acid 20 F Zeolite A 20 Ascorbic acid 3 PEG 4000 17 G Zeolite A10 Ascorbic acid 1.5 PEG 4000 8.5 H Zeolite A 7 Ascorbic acid 1 PEG 40006 ¹⁾in each case L-ascorbic acid

1. A dry microorganism culture which comprises at least onemicroorganism species in carrier-bound form, wherein the culture ispresent in the form of particles which a) have a particle size of atleast about 0.1 mm and b) comprise from about 10¹⁰ to 10¹² cfu/g of atleast one microorganism species; c) have a water activity a_(w) of lessthan 0.15: and d) are compressed.
 2. A microorganism culture as claimedin claim 1, wherein the particles have been compressed under the actionof a linear force from about 5 to 15 kN/cm or a pressure from about 90to 160 MPa.
 3. A microorganism culture as claimed in claim 1, whereinthe compressed particles comprise compacted broken material having adiameter of from about 0.1 mm to about 2 mm.
 4. A microorganism cultureas claimed in claim 1, wherein the compressed particles comprise tabletshaving a diameter of from about 2 to 50 mm and a ratio of diameter tothickness of from about 1:0.1 to about 10:1.
 5. A microorganism cultureas claimed in claim 1, wherein it comprises, a further component, aneffervescent additive.
 6. A microorganism culture as claimed in claim 1,wherein, as carrier, it comprises at least one matrix material forembedding the microorganism cells with or without at least one furthercell-stabilizing additive.
 7. A microorganism culture as claimed inclaim 1, wherein it comprises at least one lactic-acid-producingbacterial species.
 8. A microorganism culture as claimed in claim 7,wherein the bacterial species is selected from bacteria of the genusLactobacillus sp.
 9. A process for producing a dry microorganismculture, comprising at least one microorganism species in carrier-boundform and having a water activity a_(w) of less than 0.15, which processcomprises, a) dissolving or suspending at least one substance suitablefor forming a carrier in a liquid comprising at least one microorganismspecies, b) drying the resultant mixture in a spray-dryer, for thespray-drying use being made of a conditioned dried gas having a dewpoint of less than about +5° C., heated to a temperature in the range ofabove about 80° C., and c) removing the dried material from the spraydryer, this dried material having an exit temperature of from about 45to 75° C.
 10. A process as claimed in claim 9, wherein, in a furtherstage d), the dry material is subjected to a further drying at atemperature in the range from about 15 to 50° C. in a gas atmosphere orin vacuo and/or at least one desiccant is added.
 11. A process asclaimed in claim 9, wherein, as dry material, a powder concentratehaving a content of viable microorganisms of from about 5·10⁸ to 1·10¹²cfu/g is obtained.
 12. Dry compressed microorganism culture according toclaim 1, obtained from a powder concentrate of microorganism culturedried in a spray-dryer, for the spray-drying use being made of aconditioned dried gas having a dew point of less than about +5° C.,heated to a temperature in the range of above about 80° C.
 13. A processfor preparing a dry microorganism culture as claimed in claim 1, whichcomprises i) producing a powder concentrate of the microorganism cultureby carrier-bound spray-drying, carrier-bound freeze-drying or carrierbound fluidized-bed drying, ii) with or without admixing the powderconcentrate with one or more coformulants and iii compacting ortableting this mixture.
 14. A process as claimed in claim 13, whereinthe compacted powder concentrate from stage iii) is broken, with orwithout classification.
 15. A process for preparing a dry agglomeratedmicroorganism culture, which comprises i) preparing a powder concentrateof the microorganism culture by carrier-bound spray-drying, carrierbound freeze drying or carrier-bound fluidized-bed drying which powderconcentrate has a water activity a_(w) of less than 0.15, ii) with orwithout admixing the powder concentrate with one or more coformulantsand iii) agglomerating this mixture.
 16. A process as claimed in claim13, wherein the spray-drying is performed in a spray-dryer in which aconditioned dried gas is employed having a dew point of less than about+5° C., heated to a temperature in the range of above about 80° C.
 17. Astarter culture for foodstuffs and feedstuffs comprising a microorganismculture as claimed in claim 1, or prepared by a process for producing adry microorganism culture, comprising at least one microorganism speciesin carrier-bound form, which comprises a) dissolving or suspending atleast one substance suitable for forming a carrier in a liquidcomprising at least one microorganism species, b) drying the resultantmixture in a spray-dryer, for the spray-drying use being made of aconditioned dried gas having a dew point of less than about +5° C.,heated to a temperature in the range of above about 80° C., and c)removing the dried material from the spray dryer, this dried materialhaving an exit temperature of from about 45 to 75° C.
 18. A foodstuff orfeedstuff obtainable by using a microorganism culture as claimed inclaim 1 or prepared by a process for producing a dry microorganismculture, comprising at least one microorganism species in carrier-boundform, which comprises a) dissolving or suspending at least one substancesuitable for forming a carrier in a liquid comprising at least onemicroorganism species, b) drying the resultant mixture in a spray-dryer,for the spray-dryer use being made of a conditioned dried gas having adew point of less than about +5° C., heated to a temperature in therange of above about 80° C., and c) removing the dried material from thespray-dryer, this dried material having an exit temperature of fromabout 45 to 75° C.
 19. A process as claimed in claim 15, wherein thespray-drying is performed in a spray-dryer employing a conditioned driedgas having a dew point of less than about +5° C., heated to atemperature in the range of above about 80° C.
 20. A powder concentrateof a microorganism culture comprising from about 4×10¹¹ to 10¹² cfu/g ofat least one microorganism species and having a water activity a_(w) ofless than 0.15.